Probe reagent and fish using probe reagent

Abstract

Provided are: a probe reagent which is capable of stably yielding a fluorescence signal in FISH while inhibiting non-specific adsorption by the use of a nucleic acid molecule having a smaller number of bases than a BAC probe; and FISH using the probe reagent. The probe reagent is for in situ hybridization and comprises: phosphor-integrated nanoparticles containing phosphors integrated therein; and a nucleic acid molecule having a prescribed nucleic acid sequence, which phosphor-integrated nanoparticles and nucleic acid molecule are bound with each other.

The invention claimed is: 
     
       1. A probe reagent for in situ hybridization, comprising:
 a plurality of phosphor-integrated nanoparticles containing phosphors integrated therein and a matrix material, wherein said matrix material comprises a resin, and said resin is a melamine resin; and 
 a nucleic acid molecule having a prescribed nucleic acid sequence, 
 said plurality of phosphor-integrated nanoparticles and said nucleic acid molecule being bound with each other, and at least one of said plurality of phosphor-integrated nanoparticles is bound to a 3′ or 5′ terminal end of said nucleic acid molecule. 
 
     
     
       2. The probe reagent according to  claim 1 , wherein said phosphor-integrated nanoparticles have an average particle size of 300 nm or smaller. 
     
     
       3. The probe reagent according to  claim 1 , wherein said nucleic acid molecule and said phosphor-integrated nanoparticles are bound at a molar ratio of 1:1 to 1:5,000. 
     
     
       4. The probe reagent according to  claim 1 , wherein said nucleic acid molecule and said phosphor-integrated nanoparticles are bound at a molar ratio of 1:1 to 1:40. 
     
     
       5. The probe reagent for in situ hybridization according to  claim 1 , which is obtained by binding said phosphor-integrated nanoparticles containing phosphors integrated therein to said nucleic acid molecule having a prescribed nucleic acid sequence of 5,000 or less bases. 
     
     
       6. The probe reagent for in situ hybridization according to  claim 5 , which is obtained by binding said phosphor-integrated nanoparticles containing phosphors integrated therein to said nucleic acid molecule having a prescribed nucleic acid sequence of 5,000 or less bases in accordance with a method that utilizes nucleic acid terminal labeling or nick translation. 
     
     
       7. The probe reagent according to  claim 1 , wherein a labeling ratio (%) of said phosphors with respect to said nucleic acid molecule is 13.33% or less. 
     
     
       8. The probe reagent according to  claim 1 , wherein
 said phosphors integrated are low-molecular-weight fluorescent dyes or quantum dots. 
 
     
     
       9. The probe reagent according to  claim 1 , wherein
 a first biomolecule is linked to said nucleic acid molecule, 
 a second biomolecule is linked to said phosphor-integrated nanoparticles, and 
 said nucleic acid molecule and said phosphor-integrated nanoparticles are bound via specific bonds formed by said first and second biomolecules. 
 
     
     
       10. The probe reagent according to  claim 9 , wherein said first and second biomolecules are each streptavidin or biotin. 
     
     
       11. The probe reagent according to  claim 1 , wherein said phosphor-integrated nanoparticles are directly bound to a base of said nucleic acid molecule. 
     
     
       12. The probe reagent according to  claim 11 , wherein said phosphor-integrated nanoparticles are covalently-bound to the base of said nucleic acid molecule. 
     
     
       13. The probe reagent according to  claim 1 , wherein the phosphors are dispersed inside the nanoparticles or bound to surfaces of the nanoparticles. 
     
     
       14. The probe reagent according to  claim 1 , wherein the nucleic acid molecule has a nucleic acid sequence of 5,000 or less bases. 
     
     
       15. A probe reagent for in situ hybridization, comprising:
 a plurality of fluorescent nanoparticles containing phosphors integrated therein and a matrix material, wherein said matrix material comprises a resin, and said resin is a melamine resin; and 
 a nucleic acid molecule having a prescribed nucleic acid sequence, 
 wherein the plurality of fluorescent nanoparticles is bound to the nucleic acid molecule, at least one of said plurality of fluorescent nanoparticles is bound to a 3′ or 5′ terminal end of said nucleic acid molecule, and 20,000 mol or more of the plurality of fluorescent nanoparticles are bound per 1 mol of a nucleic acid molecule. 
 
     
     
       16. A probe reagent kit for FISH, separately comprising:
 a nucleic acid molecule which has a sequence of less than 150,000 bases that is complementary to a sequence of a specific region on a chromosome and to which a first biomolecule is linked; and 
 a plurality of phosphor-integrated nanoparticles to which a second biomolecule capable of specifically binding to said first biomolecule is linked; 
 wherein said plurality phosphor-integrated nanoparticles contain phosphors integrated therein and a matrix material, wherein said matrix material comprises a resin, and said resin is a melamine resin, and 
 said plurality of phosphor-integrated nanoparticles and said nucleic acid molecule are configured to be bound with each other, and at least one of said plurality of phosphor-integrated nanoparticles is configured to be bound to a 3′ or 5′ terminal end of said nucleic acid molecule. 
 
     
     
       17. A probe reagent kit for FISH, separately comprising:
 a nucleic acid molecule which has a sequence of 5,000 or less bases that is complementary to a sequence of a specific region on a chromosome and to which a first biomolecule is linked to a terminal(s) and/or 1 to 50 spots other than said terminals; and 
 a plurality of phosphor-integrated nanoparticles to which a second biomolecule capable of specifically binding to said first biomolecule is linked; 
 wherein said plurality phosphor-integrated nanoparticles contain phosphors integrated therein and a matrix material, wherein said matrix material comprises a resin, and said resin is a melamine resin, and 
 said plurality of phosphor-integrated nanoparticles and said nucleic acid molecule are configured to be bound with each other, and at least one of said plurality of phosphor-integrated nanoparticles is configured to be bound to a 3′ or 5′ terminal end of said nucleic acid molecule. 
 
     
     
       18. A method for FISH using the probe reagent according to  claim 1 .